The overall goal of this project is to understand the pathobiology of Aspergillus fumigatus which is one of the most serious fungal pathogens that causes life-threatening systemic disease mostly in patients with neutropenia or defective phagocytic cells. The primary etiologic agent of invasive aspergillosis is Aspergillus fumigatus, a ubiquitous environmental fungus. By using molecular methods, we chose to identify the traits that are associated with virulence of the species. We are particularly interested in the factors that enable the fungus to ward off the host defense system. We use neutrophils from normal as well as CGD patients and study the host-pathogen interaction in order to determine the fungus specific genes that are essential for survival of the fungus during such interactions. We also use a murine systemic infection model to identify the fungal factors that are required for the production of fatal disease. We are also aiming to find new antifungal agent that will be efficacious for the Aspergillus species that cause infection refractory to existing antifungal antibiotics. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease(CGD) of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. During 2007-2008, we investigated the Aspergillus response to host immune cells by studying the genes differentially expressed in conidia vs hyphae when challenged with neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD). To our knowledge, this is the first study that investigated the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with CGD which are defective in the production of reactive oxygen species. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants of several of these differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed a higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack. During 2008-2009, we characterized the differences in disease manifestations and the manner of dissemination beween A. fumigatus and its sister species, A. udagawae. Both A. udagawae (Neosartorya udagawae) and A. fumigatus belong to Section Fumigatii in the genus Aspergillus and they cannot be differentiated by their morphological characteristics but can be differentiated by genetic methods. We also characterized the biological differences between the two species. During the 2009-2010, we focused on the identification of MAT-1 and MAT-2 strains that could be used as the foundational strains for the construction of an isogenic pair for the genetic study of virulence factors and other traits of pathobiological importance. We found two super maters of the opposite mating type and decided to use the AFB62 strain that originated from a case of invasive aspergillosis. During 2010-2011, we characterized the sexual reproduction by the super mater pair which was chosen for the construction of the MAT-1 and MAT-2 isogenic set. The meiotic product (ascospores) of the pair could be readily purified from contaminating mitotic spores by heat treatment for 30 minutes at 70 C. High recombination frequencies obtained by mating two conidial color mutants derived from the super mater pair:MAT-1 carrying abr2 mutation, and MAT-2 carrying alb1 mutation and the mutated genes were located 19 kb apart on the same chromosome, indicated that the pair can be used as a genetic tool for recombinational analysis. During 2011-2012, we discovered a new pathogenic species of Aspergillus phylogenetically unrelated to A. fumigatus that causes invasive aspergillosis refractory to currently available antimycotic agents. The new species was described as A. tanneri belonging to the Aspergillus section Circumdati. The species was innately resistant to polyenes, azoles and echinocandins and caused fatal infections in CGD patients. During 2012-2013, we compared the virulence of A.tanneri with that of A. fumigatus in various host systems including CGD mice, corticosteroid treated mice and Galleria mellonela and found that the new species is significantly more virulent for Galleria but less virulent for the mice models. During 2013-2014, we discovered three new species A. fumigatus-look alike species that are genetically and pathobiologically different from A. fumigatus. During 2014-2015, we have identified new drug resistance gene using the whole genome sequence of the isogenic mating pair combined with creating drug resistant mutants. During 2015-2016, we found that exogenous Type 1 IFN protects mice infected with A. fumigatus as well as the Aspergillus species resistant to antifungals such as A. tanneri, A. nidulans. During 2016-2017, we constructed bioluminescent strains of A. nidulans and A. fumigatus to compare the mode of dissemination in the two species. We also discovered that a new orotomide compound which inhibits dihydroorotate dehydrogenase protects mice from aspergillosis caused by A. nidulans and its efficacy is being tested for aspergillosis caused by A. tanneri. During 2017-2018, we found that Olorofim (formal name of orotomide drug) protects mice from all three species of Aspergillus including two species refractory to currently available antifungal drugs.